Distinct mechanisms of site-specific DNA damage induced by endogenous reductants in the presence of iron(III) and copper(II)

The ability of Cu(II) and Fe(III) to promote site-specific DNA damage in the presence of endogenous reductants was investigated by using P-32-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Ascorbate induced metal-dependent DNA damage most efficiently (ascorbate > GSH > NADH). Cu(II) induced endogenous reductants-dependent DNA damage more efficiently than Fe(III). Endogenous reductants plus Fe(III) caused DNA cleavage at every nucleotide, without marked site preference. DNA damage by Fe(III) was inhibited by hydroxyl free radical ((OH)-O-.) scavengers and catalase. These results suggest that endogenous reductants plus Fe(III) generate free or extremely near free (OH)-O-. via H2O2 formation, and that (OH)-O-. causes DNA damage. In the presence of 50 mu M Cu(II) in bicarbonate buffer, ascorbate caused DNA cleavage frequently at sites of two or more adjacent guanine residues. In contrast, in the presence of 20 mu M Cu(II), ascorbate caused DNA cleavage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited DNA damage by Cu(II), whereas (OH)-O-. scavengers did not. Fe(III)-dependent 8-oxo-7,8-dihydro-2'-deoxyguanosine formation was inhibited by (OH)-O-. scavengers, whereas no inhibition by (OH)-O-. scavengers was observed with Cu(II). These results suggest that (OH)-O-. is the main active species formed with Fe(III), whereas copper-peroxide complexes with a reactivity similar to (OH)-O-. participate in Cu(II)-dependent DNA damage. The polyguanosine sequence specificity of DNA damage in the presence of high concentrations of Cu(II) can be explained by the preferential binding of Cu(II) to guanine residues. (C) 1998 Elsevier Science B.V. All rights reserved.